The goal of this presentation is to share recent developments in the analytical approaches to detect, quantify, and differentiate impurities in biologics that are the result of protein-container interaction and aggregation. 

In protein-based formulations, distinguishing aggregated API from other particle types is important for understanding the root cause of instability. Until now, existing methods have been either unreliable or too cumbersome and difficult to use in many workflows. Here we introduce the Aura, a 96-well low-volume aggregate and particle imaging system that can rapidly size, count, and characterize particles and identify them as proteins, non-proteins, hydrophobic, or other types of molecules.

Low Endotoxin Recovery (LER) is a phenomenon that can occur during LAL compendial tests. This is sometimes caused by components in the formulation that mask the endotoxin in the sample. Due to safety issues that can be caused by LER, the FDA’s Center for Drug Evaluation and Research requests that LER studies be conducted. Study design and the appropriate stage of development to perform an LER study will be discussed.  

The use of kD and B22 in predicting the PPI in concentrated mAb formulations was evaluated through comparison with S(q)eff extracted from SAXS/SANS measurements. The disagreements between PPI determined from dilute (kD/B22) and concentrated solutions (S(q)eff) highlight the necessities of performing measurements directly from concentrated mAb solutions. Also, the correlation between the measured and predicted viscosity results suggests a better understanding of the relationship between PPI and solution viscosity is needed so that more reliable predictions can be made from PPI information.

High Molecular Weight Species (HMWS) is important to monitor and control due to its potential impact on efficacy and safety. Some HMWS could reverse soon after administration to patients. The dissociation of HMWS reduces its exposure in patients, thereby mitigating potential HMWS associated risk. Here, we developed an in vitro model system to study reversibility of HMWS in a biologically relevant environment that mimics in vivo context.

The structural integrity of polysorbate (PS) is important for its function as a surfactant to protect proteins from aggregation. We have explored the role of light-induced, protein-derived radicals on the cis/trans isomerization of unsaturated fatty acids in PS80. A mechanistic study performed with a combination of N-acetyltryptophan amide and glutathione disulfide suggested the involvement of thiyl radicals, generated by photoinduced electron transfer from Trp to the disulfide, in cis/trans isomerization.

Polysorbate 20 and 80 are essential excipients to stabilize biopharmaceutical formulations. However, polysorbate is prone to degradation induced by (enzymatic) hydrolysis and/or oxidation. An overview on degradation pathways and analytical tools for polysorbate (focus on LC-CAD and LC-MS) will be given. For LC-MS analysis, novel universal markers for oxidation will be presented, as well as a novel hypothesis on the role of micelles for polysorbate oxidation.

Polysorbate 80 is a commonly used excipient for multiple Sanofi biotherapeutics, including enzyme replacement therapies (ERTs), antibodies, antibody drug conjugates (ADCs), and gene therapies, to ensure their stability. A minimal concentration of PS80 is required to maintain its effectiveness for preventing aggregation, and unwanted degradation leads to a decrease of PS80 concentration and particle formation in drug substance and drug product.  Here we present several hyphenated chromatographic methods recently developed in analytical development for characterization of PS80, including HPLC-CAD, 2D HPLC-CAD, and UPLC-QDa. We demonstrate that these methods can be used to quantitatively and qualitatively determine the PS80 content and investigate the degradation pathways, to support product understanding and formulation development.